Spalax fibroblast-derived anti-cancer agents

ABSTRACT

A conditioned cell culture medium of cells derived from Spalax or naked mole rat (Heterocephalus glaber) and methods for preparing it are provided. Pharmaceutical compositions comprising the conditioned cell culture medium and its use in the treatment of cancer as well as methods for identifying anti-cancer agents are also provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority from U.S. provisional patentapplication No. 61/751,051 filed on Jan. 10, 2013, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of cancer treatment and inparticular to compositions comprising Spalax derived anti-cancer agents.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the world.Notwithstanding steady progress in the understanding of thismultifaceted disease, many cancers still are not treatable. Laboratorymice and rats provided invaluable knowledge in biomedical research andpharmaceutics; however, these animals were subjected to inbreeding andartificial selection for the experiments' standardization purposes,which ultimately caused loss of stress tolerance and naturally selectedfeatures.

Throughout the last forty years, several thousand Spalax individualshave been housed and studied in the Animal Facility at the Institute ofEvolution of Haifa University. Despite this small rodent's (˜100-200gr.) extremely long lifespan (>20 years), none of the animals have everdeveloped spontaneous tumors, nor do they show any aging-relatedphenotypic changes. The mole rat, Spalax ehrenbergi, is a wild, solitaryrodent of the Eastern Mediterranean region. Spalax inhabits a system ofpoorly ventilated dark, sealed underground tunnels protected fromclimatic extremes, pathogens, and predation. During the Mediterraneanrainy season animals are engaged in intensive digging under extremehypoxic conditions. Spalax has evolved a unique adaptive complex forliving underground, including a unique ability to cope with extremehypoxia and hypercapnia (Nevo et al, 2001). Spalax can conduct intensiveaerobic work under low O₂ pressures (down to 3% O₂) due to increasedmuscular mass, high density of blood vessels and mitochondria resultingin reduced oxygen diffusion distance and efficient oxygen delivery evenat low capillary PO₂ (Nevo et al. 2001; Shams et al. 2005a). Hypoxia canresult in a failure to maintain essential cellular functions andcontributes to cardio- and cerebrovascular failure, pulmonary diseasesand cancer, which together are the primary sources of morbidity in thewestern world. Spalax genes exhibit hypoxia-related adaptations instructure and function (Shams et al. 2005b; Ravid et al. 2007; Avivi etal. 2010; Schulke et al. 2012). Noteworthy are VEGF, constitutivelyhighly expressed as compared to rats (Avivi et al. 2005); p53 thatharbors mutations in the DNA-binding site, identical to the most commonp53 mutations in tumors, however, in Spalax renders bias againstapoptosis but favors cell cycle arrest/DNA repair both in vitro and invivo (Avivi et al. 2007); and a unique Spalax heparanase splice variantthat was shown to decrease tumor size by a factor of 7 as well asreduced metastatic activity compared to native heparanase (Nasser et al.2009). Furthermore, assessment of Spalax transcriptome assembly data hasrevealed enrichment of genes that overlap cancer-resistance, apoptosis,angiogenesis pathways and hypoxia-tolerance (Malik et al. 2011; Malik etal. 2012). This provides evidence that Spalax are extraordinarily highlyresistant to malignant transformation. Elucidating the mechanismsemployed by this wild non inbred, naturally cancer-resistant Spalax hasgreat importance as early curing and preventative measures may be themost efficient way of dealing with increased cancer rates. It is anobject of the present invention to provide anti-cancer agents based onthe intrinsic mechanisms of Spalax and related species.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a conditioned cellculture medium of cells derived from Spalax or naked mole rat(Heterocephalus glaber), or a biologically active fraction thereof.

In another aspect, the present invention provides a method foridentifying an anti-cancer agent, comprising: (a) obtaining aconditioned cell culture medium of cells as defined herein below; (b)fractionating said conditioned cell culture medium, thereby obtainingfractions; (c) contacting cancer cells and normal cells with saidfractions of (b), and identifying active fractions that kill or inhibitthe proliferation of cancer cells but have no or little effect onproliferation of normal cells; and (d) collecting said active fractionsof (c) and optionally repeating step (c) until one or more anti-canceragent(s) are identified.

In an additional aspect, the present invention is directed to a methodfor producing a conditioned cell culture medium of cells derived fromSpalax or naked mole rat (Heterocephalus glaber), comprising: (a)obtaining Spalax or naked mole rat fibroblasts; (b) culturing saidSpalax or naked mole rat fibroblasts in a growth medium, therebyproducing a Spalax or naked mole rat fibroblast-conditioned composition;and (c) collecting said Spalax or naked mole rat fibroblast-conditionedcomposition.

In still another aspect, the present invention provides an active agentselected from: (i) a conditioned cell culture medium of cells derivedfrom Spalax or naked mole rat (Heterocephalus glaber); (ii) abiologically active fraction of (i); or (iii) a pharmaceuticalcomposition comprising (i) or (ii) and a pharmaceutically activecarrier, excipient or diluent.

In yet another aspect, the present invention relates to a method fortreatment of cancer or inhibiting cancer metastasis in a subjectdiagnosed with cancer, said method comprising administering to saidsubject an effective amount of the active agent of the presentinvention.

In still an additional aspect, the present invention provides a methodfor killing cancer cells, inhibiting proliferation of cancer cells orinhibiting cancer cell migration, comprising administering to a subjectin need thereof an effective amount of the active agent of the presentinvention,

In yet an additional aspect, the present invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier, excipient or diluent and the active agent of the presentinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A and FIG. 1B show the effect of DMBA/TPA applications on skincancer induction in Spalax and mice: Representative images showingmacroscopic and microscopic skin changes in Spalax (FIG. 1A) and mice(FIG. 1B). (FIG. 1A) Normal pre-treatment tissues (left image).Extensive necrosis of skin and subcutaneous adipose tissue were foundafter 10 days (middle image). Completely healed skin lesion showingepidermal thickening with hyperkeratosis and prominent dermal fibrosis(right image). Hematoxylin and eosin staining, ×40 (left and middleimages) and ×100 (right image). (FIG. 1B) Normal pretreatment tissues(left images). Intra-epidermal blisters, partially ruptured with erosionformation and crusting, congestion, and inflammatory cell infiltratewithin the dermis indicate ongoing inflammation (middle image). Skinpapillary outgrowths with thickened, dysplastic epidermis, numerousmitoses and foci suggestive of invasive squamous cell carcinoma.Hematoxylin and eosin staining, ×40 (right images).

FIG. 2 depicts the effect of 3-Methylcholantren treatment on soft tissuetumor induction in Spalax and mice. Animals were treated with a singleinjection of 3MCA as follows: 200 μg/200 μL for mice; 1 mg/500 μL forSpalax. Animals were observed once a week until development ofpathological process, (tumors could be palpated), and then 2-3 times aweek. Animals were sacrificed, tissues were removed, and eitherimmediately frozen in Liquid-N₂ and kept at −80° C., or fixed inparaformaldehyde. Representative images show macroscopic and microscopicobservations. Mouse: An ill-defined, soft mass, with foci of necrosisand hemorrhage; diagnosed as high-grade fibrosarcoma by histology.Spalax: a well-circumscribed, firm, whitish nodule composed of benignspindle cells organized into long regular bundles—benign reactivefibrosis.

FIG. 3A-FIG. 3D depict 3MCA-induced fibrosarcoma in Spalax. (FIG. 3A)Pathologic specimen, toluidine blue staining. Note spindle shaped cells;nuclei are variable in shape, size and chromatin distribution. Nucleolivary in frequency. Gant cells are present. (FIG. 3B) Transmissionelectron microscopy (TEM): Dilated, elongated rough endoplasmicreticulum (black arrow) and collagen fibers (white arrow) (FIG. 3C) TEM:giant, monstrous nucleus (N). (FIG. 3D) Cell line established fromSpalax fibrosarcomas, phase contrast image after 6 months of continuouscultivation (×20).

FIG. 4 shows effect of co-culture of fibroblasts with Hep3B cancercells. Spalax fibroblasts kill co-cultured cancer cells. Hep3B cells(TC) were cultured in Roswell Park Memorial Institute medium(RPMI)/Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) (1:1) supplemented with 15% fetal bovine serum (HIS), alone(left column, or co-cultured with mouse newborn normal fibroblasts (MF)(middle column), and with Spalax newborn normal fibroblasts (rightcolumn). Fibroblasts were plated first (5×10⁴, in 6-well plates), andcancer cells were added within 1 h (5×10³), with a 10:1fibroblast-to-cancer cell ratio. In parallel, control cultures of cancercells and fibroblasts were plated with the same number of cellsseparately. White arrows point to the foci of destroyed cancer cells,and black arrows show the fibroblast -tumor cell colony boundaries.Cells in mono- and co-cultures were observed and photographed daily.Representative images of eight plates for each sample at different timeintervals are shown. Magnification ×200

FIG. 5 shows the effects of Spalax lung and Acomys skin fibroblasts ongrowth of co-cultured human hepatoma cells (Hep3B). Cancer cells werecultured either alone or in presence of Acomys and Spalax lungfibroblasts in the ratio of 1:10 (5×104 fibroblasts and 5×103 cancercells in 6-well plates) in RPMI/DMEM-F12 media. (1:1) containing 10%FBS. The phase contrast images (×200) represent cells interactions after6, 11, and 14 co-culturing days. Black arrows point to fibroblastssurrounding cancer cell colonies. White arrows show foci of dead cancercells.

FIG. 6A-FIG. 6F show suppressing effects of Spalax fibroblastsconditioned media on different cancer cells of various origins. (FIG.6A) Hep3B cells were seeded in a 96-well plate at a density of 5×10³ and1×10³ cells/well in RPMI-DMEM/F12 culture medium supplemented with 10%FBS, conditioned with Spalax or mouse skin newborn fibroblasts or nofibroblast control. Hep3B cells were incubated for 4 days; levels ofproliferation were estimated by Presto Blue® Reagent. Results arepresented as percentage of control, mean±S.D. (FIG. 6B) Spalaxfibrosarcoma cells (SpFS2240) were incubated for 3 or 7 days in fullmedium or under conditioned medium (CM) of Spalax adult skin normalfibroblasts (SpSNF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240CM). Cell viability was evaluated by using Presto Blue® reagent. Resultsare presented as percentage of control (SpFS2240 CM), mean±S.D. (FIG.6C) MCF7 cells (5×10³ cells) were grown in soft agar on top of amonolayer of mouse newborn (MNbF), or Spalax newborn (SpNbF) fibroblastsin 35-mm culture dishes. Dishes were incubated until cell colonies werewell defined. Colonies were stained with 0.05% crystal violet solutionand counted under light microscope after 5 and 11 days in soft agar.(FIG. 6D, FIG. 6E) Hep3B cells (1×10⁴ cell/well) were cultured in 6-wellplates under conditioned medium of Spalax adult skin fibroblasts (FIG.6D) or grown in medium generated by Hep3B cells (FIG. 6E). After 9-days,cells were collected, and their survival rates were assessed by trepanblue extrusion assay and analyzed by using an automatic cell counter(Countess®, Life Technologies).Viable and dead cell size and numbers arepresented in black and white columns, respectively (FIG. 6F) Effects ofconditioned media generated by Spalax normal fibroblasts on growth ofnormal fibroblasts. The levels of viability were estimated after 4 daysby Presto Blue® reagent, mean±S.D. All results were obtained from threeindependent experiments performed in 3-6 technical repeats.

FIG. 7 shows that naked mole rat (Heterocephalus) fibroblasts restrictthe growth and kill cancer cells in co-culture experiments. Hep3B tumorcells were cultured either alone or in presence of Heterocephalusfibroblasts in the ratio of 1:10 (5×10⁴ fibroblasts and 5×10³ cancercells in six-well plates) in RPMI/DMEM-F12 media (1:1) containing 10%FBS. After seven days incubation cells were photographed.Microphotographic images are shown (×200). White arrows point to thefoci of damaged cancer cells. Black arrows point to fibroblastssurrounding cancer cells colonies TC, tumor cells.

FIG. 8 shows that Spalax normal fibroblast-conditioned medium, but notimmortalized cells CM, compromises cell cycle, of Hep3B cells. Hep3Bcells were grown on cover slips under medium conditioned by Spalaxnormal fibroblasts (middle chart) or Spalax immortalized fibroblasts(right chart) for seven days. Cells were harvested and stained withpropidium iodide, and cell cycles were analyzed by flow cytometry.Spalax immortalized (by serial passages) cells lost their ability toinhibit cancer cells.

FIG. 9 depicts the effects of Spalax normal and immortal fibroblasts onthe migration of breast cancer cells MDA-MB-231. Fibroblasts were seededinto 24-well plates (Transwelt®, Corning Inc.) at the density of 10⁵cells/well, incubated 48 h, thereafter the inserts containing 25×10MDA-MB-231 were immersed into lower chambers without changing medium. 24hours after the incubated membranes were fixed with 2.5% glutaraldehyde(GA) for 10 min, washed in double-distilled water (DDW), and stainedwith 0.5% toluidine blue for 5 min and photographed. Color intensityreflects the cells that crossed to the lower of insert (migratingcells).

DETAILED DESCRIPTION

It has been found in accordance with the present invention that Spalaxis resistant to two-stage DMBA/TRA, and 3-MCA carcinogens (Manov et al.2013). DMBA/TPA is commonly used to study malignant transformationresembling formation of human squamous cell carcinoma (Yuspa 1998). In asingle dose, DMBA has been shown to induce substantial oxidative stress(Izzotti et al. 1999), followed by repetitive application of TPA thatcause persistent inflammation supporting tumorigenesis (Goerttler et al.1984). Example 1 herein below shows that mice treated by DMBA/TPAinitially developed benign papillomas, which subsequently transformed tosquamous cell carcinomas. In contrast, treatment of Spalax led tonecrotic wounds which completely healed with no signs of malignancy. Thecarcinogen 3-MCA is known to produce primary skin fibrosarcoma throughpersistent inflammation leading to DNA-adducts (Krelin et al. 2007).3-MCA is metabolized via P450 enzymes to form a reactive metabolitecausing severe oxidative damage (Flesher et al. 1998). In our study,100% of 3-MCA-injected mice and rats developed tumors at the injectionsite within 2-3 and 4-6 months, respectively. Following the first yearof treatment no Spalax animals showed any pathological process. Howevertwo individuals out of eight developed benign fibrotic overgrowth after14 and 16 months respectively, and only one case of malignanttransformation in a >10 years-old Spalax animal was recognized 18 monthafter 3-MCA injection.

The present inventors developed a co-culture system involving normalprimary fibroblasts isolated from different rodent species (Spalax,naked mole rat Heterocephalus glaber, mouse, and spiny mice Acomyscahirinus), with human hepatocellular and breast carcinomas (Hep3B andMCF7 cells), as well as Spalax- derived fibrosarcoma cells (SpFS2240).It has been found in accordance with the present invention (Example 2)that both Spalax and Heterocephalus fibroblasts restrict malignantbehavior either through direct fibroblast-cancer cell interaction or viasoluble factors secreted by normal Spalax fibroblasts into conditionedmedium.

The present invention thus relates, in one aspect, to isolatedfibroblasts derived from Spalax or naked mole rat (Heterocephalusglaber) for use in producing a Spalax fibroblast-conditioned compositionor naked mole rat fibroblast-conditioned composition.

In another aspect, the present invention provides a conditioned cellculture medium of cells derived from Spalax or naked mole rat(Heterocephalus glaber), or a biologically active fraction thereof.

The term “conditioned medium” in general refers to a growth medium inwhich cells have been cultured/incubated for a period of time followedby harvest of such medium from the cultured cells. A conditioned mediumcontains metabolites, growth factors, and extracellular matrix proteinssecreted into the medium by the cultured cells.

In accordance with the present invention, the term “conditioned mediumof cells derived from Spalax or naked mole rat (Heterocephalus glaber)”refers to the medium harvested from the cultured cells that wereoriginally isolated from these animals. The conditioned medium may hederived from a medium suitable for culture of Spalax or naked mole ratcells such as, but not limited to, DMEM-F12 medium, and may containadditional nutrients. The suitable culture medium is easily identifiedby culturing the cells in the desired medium and identifying healthypropagation or absence of senescence of the cells. The terms “Spalaxcell-conditioned composition” and “naked mole rat cell-conditionedcomposition” are used interchangeably herein with the terms “conditionedmedium of cells derived from Spalax” and “conditioned medium of cellsderived from naked mole rat (Heterocephalus glaber)”, respectively.

Thus, in certain embodiments, the cells are derived from Nannospalaxehrenbergi (Spalax ehrenbergi). In certain embodiments the cells areselected from the group consisting of adipocytes, lymphatic cell,endothelial cells, hepatocytes and intestinal cells, kidney epithelialcells, placental epithelial and endothelial cells, and in particular,the cells are skin or lung fibroblasts.

In certain embodiments, the cells, such as the fibroblasts, are primarycells, i.e. they are harvested from living animals and cultured untilthey stop dividing and senesce.

In certain embodiments, the fibroblasts form an immortalized cell line.

As used herein, the term “a biologically active fraction thereof” refersto a fraction of the conditioned cell culture medium of cells derivedfrom Spalax or naked mole rat obtained by fractionation of theconditioned medium and shown to retain the same biological activities asshown herein for the whole conditioned medium such as cytotoxic oranti-metastatic activity. Conveniently, the biological activity may bedetermined using a xenograft, e.g. but not limited to transplantation ofhuman cancer cells in a mouse, or in vitro by utilizing culture wellsand cell attachment substrates, such as, but not limited to, porousmembranes (such as Transwell®, Corning Inc.), that allow observation ofcell migration in the presence or absence of certain cells or activeagents. The fractionation of the conditioned medium may be carried outby standard procedures, e.g. organic extractions, size exclusionfractions and/or reverse phase chromatography carried out using e.g.high pressure liquid chromatography (HPLC) columns and pumps, as knownin the art.

In another aspect, the present invention provides a method foridentifying at least one anti-cancer agent, comprising: (a) obtaining aconditioned cell culture medium of cells as defined herein above; (b)fractionating said conditioned cell culture medium, thereby obtainingfractions; (c) contacting cancer cells and normal cells with saidfractions of (b), and identifying active fractions that kill or inhibitthe proliferation of cancer cells but have no or little effect onproliferation of normal cells; and (d) collecting said active fractionsof (c) and optionally repeating step (c) until one or more anti-canceragent(s) are identified.

The optional step of repeating step (c) refers to the repeatedfractionation of fractions obtained in step (d) using other techniquesin order to separate various compounds collected in the same fraction.

In certain embodiments, the cancer and normal cells used in the methodfor identifying the at least one anti-cancer agent are human cells,which may be epithelial cells and mesenchymal cells, respectively.

In an additional aspect, the present invention is directed to a methodfor producing a conditioned cell culture medium of cells derived fromSpalax or naked mole rat (Heterocephalus glaber) comprising: (a)obtaining ,Spalax or naked mole rat cells; (b) culturing said Spalax ornaked mole rat cells in a growth medium, thereby, producing a Spalax ornaked mole rat cell-conditioned composition; and (c) collecting saidSpalax or naked mole rat cell-conditioned composition.

In certain embodiments, the cells utilized for making the Spalax ornaked mole rat cell-conditioned composition are selected from the groupconsisting of adipocytes, lymphatic cells, endothelial cells,hepatocytes and intestinal cells, kidney epithelial cells, placentalepithelial and endothelial cells, and in particular, the cells are skinor lung fibroblasts.

The procedure for obtaining Spalax or naked mole rat cells is well-knownin the art and may involve partial digestion of animal tissues bycollagenase, followed by multiple washing procedures by using culturemedia supplemented with fetal bovine serum, and plating in tissueculture plates containing antibiotic and antimycotic agents. Theconditioned medium may be derived from a medium suitable for culture ofSpalax or naked mole rat cells such as, but not limited to, DMEM-F12medium, and may contain additional nutrients. The collection of theconditioned medium may be done by, for example, but not limited to,centrifugation of the cells and the collection of the supernatant or byfiltering the cells and collecting the filtrate.

In still another aspect, the present invention provides an active agentselected from: (i) a conditioned cell culture medium of cells derivedfrom Spalax or naked mole rat (Heterocephalus glaber); (ii) abiologically active fraction of (i); or (iii) a pharmaceuticalcomposition comprising (i) or (ii) and a pharmaceutically activecarrier, excipient or diluent.

In yet another aspect, the present invention relates to a method fortreatment of cancer or inhibiting cancer metastasis in a subjectdiagnosed with cancer, said method comprising administering to saidsubject an effective amount of the active agent of the presentinvention.

In still an additional aspect, the present invention provides a methodfor killing cancer cells, inhibiting proliferation of cancer cells orinhibiting cancer cell migration, comprising administering to a subjectin need thereof an effective amount of the present invention.

The term “treating” or “treatment” as used herein includes abrogating,substantially inhibiting, slowing or reversing the progression of acondition, substantially ameliorating clinical symptoms of a conditionor substantially preventing the appearance of clinical symptoms of acondition. With regard to cancer, the term refers to preventing ordelaying cancer, inhibiting tumor growth or causing death of cancercells, including primary and metastatic cancer cells. Such treatment canalso lead to regression of tumor growth, i.e., to decrease in size orcomplete regression of the tumor, and to elimination of metastases. Theterms “tumor” and “cancer” are used interchangeably herein.

In certain embodiments, the cancer cells are selected from the groupconsisting of carcinoma cells, sarcoma cells, glioma cells, leukemiacells and lymphoma cells. In particular, the carcinoma cells may beselected from the group consisting of breast cancer carcinoma cells,hepatocellular carcinoma cells and breast adenocarcinoma cells.

In some embodiments of the invention, the treatment of cancer comprisesinhibiting cancer metastasis.

In yet an additional aspect, the present invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier, excipient or diluent and the active agent of the presentinvention.

Optionally, the active agents or compositions of the invention may beadministered to the subject in combination (concurrently orsequentially) with other anti-cancer agents or treatments. For example,they may be administered in combination with one or morechemotherapeutic agents such as, but not limited to, alkylating agents,e.g. Cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, Carmustine(BCNU, Gliadel), Lomustine (CCNU), Decarbazine, Procarbazine, Busulfan,and Thiotepa; antimetabolites, e.g. Methotraxate, 5-Fluorouracil,Cytarabine, Gemcitabine, 6-mercaptopurine, 6-thioguanine, Fludarabine,and Cladribine; anthracyclins, e.g. daunorubicin. Doxorubicin,Idarubicin, Epirubicin and Mitoxantrone; camptothecins, e.g. irinotecanand topotecan; taxanes, e.g. paclitaxel and docetaxel; and platinums,e,g. Cisplatin, carboplatin, and Oxaliplatin, as well as toimmunotherapies, e.g, Herceptin and Cetuximab, hormone responsivetherapies, e.g. Tamoxifen, Raloxifene, Fulvestrant, Anastrozole,Letrozole or Exemestane for breast cancer, or anti-androgens e.g.flutamide for prostate cancer, small molecules inhibiting epidermalgrowth factor receptor (EGFR, e.g. Lapatinib or gefitinib),anti-angiogenic therapy, e.g. Bevacizumab, sunitinib, sorafenib andpazopanib, antibodies and small molecules targeted against beta 1integrins (e.g. ATN-161, Volociximab and JSM6427), or inhibitors,antagonists and small molecules against urokinase receptor (UPAR).

Advantageously, the compositions of the invention may be used inconjunction (concurrently or sequentially) with surgery or radiotherapy.For example, the active agent of the invention may be used concomitantlywith, or within 1-4 days of, a surgical treatment for cancer.Dissemination of tumor cells, which are the source for the progressionto metastatic disease, may occur as a result of the surgical operation.By performing such procedures in conjunction with the compositions ofthe invention, cancer metastasis may be prevented or inhibited. Thus thecompositions and methods of the invention may be used to inhibitpost-surgery metastatic process.

Given that dissemination of tumor cells may have already occurred evenat an early stage of tumor progression, the anti-cancer agents ortreatments that may be administered in combination with the compositionsof the invention, include, in some embodiments, neoadjuvant treatment,namely radiotherapy, chemotherapy, hormone therapy and/or immunotherapyused for shrinking the size of the tumor prior to surgical operation.

The active agent of the present invention or fractions thereof can beadministered to individuals in need per se or in a pharmaceuticalcomposition with suitable carriers, excipients or diluents.

The term “pharmaceutically acceptable carrier” refers to a carrier ordiluent that does not cause irritation or other adverse effect to anorganism and does not have an adverse effect on the biological activityand properties of the administered compound. The “excipient” refers toan inert substance added to a pharmaceutical composition to furtherfacilitate administration of an active ingredient. Examples, withoutlimitation, of excipients include calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils and polyethylene glycols.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active agents intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal or intraocular injections.

Alternatively, the pharmaceutical composition may be administered in alocal rather than systemic manner, for example, via injection directlyinto a tissue region of a patient.

For injection, the active ingredients of the compositions of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants for example DMSO, orpolyethylene glycol are generally known in the art.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a “therapeutically effective amount” means an amount of anactive ingredient effective to prevent, alleviate or ameliorate symptomsof a disease or disorder or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations ora single administration of a slow release composition, with course oftreatment lasting from several days to several weeks or until cure iseffected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

The present invention further encompasses an isolated Spalax cancer cellline. In certain embodiments, the isolated Spalax cancer cell line isestablished from Spalax fibrosarcoma, in particular it may beestablished from Spalax ehrenbogi (Nannospalax) fibrosarcoma, forexample, as in the specific case described herein below in Example 1, itmay be a isolated Spalax cancer cell line designated SpFS2240.

The present invention will now be described in more detail in thefollowing non-limiting Examples and the accompanying figures.

EXAMPLES Materials and Methods

Animals: Blind mole-rat (Spalax), spiny mice (Acomys cahirinus), rats(Rattus norvegicus), and C57BL/6 mice were subjected to DMPA/TPA or 3MCAtreatments. For DMBA/TPA treatment, eight Spalax and six rr3iceindividuals were used. For 3MCA treatment 12 Spalax, six mice, and sixrats were used.

DMBA/TPA treatment: A single application of 200 μg of DMBA dissolved in100 μL of acetone for mice, and 500 μg in 250 μL for Spalax were used.Three days thereafter, mice and Spalax were treated with 30- or 60 μg ofTPA dissolved in 100- or 250 μL of acetone, respectively, three timesper week for 2-3 months, until all mice developed advanced cancer andwere subsequently sacrificed. Spalax continued to be treated for anadditional three months.

3-MCA carcinogen treatment: Animals were treated with a singlesubcutaneous injection of 3MCA dissolved in olive oil in the upper backas follows: 200 μg/200 μL for mice; 1 mg/500 μL for Spalax; and 1.5mg/500 μL for rats (according to average body weight).

Cell culture: Primary fibroblasts were isolated from skin and lungs ofSpalax, naked mole rat, mice, and Acomys as described in SI (Materialsand Methods). Human cancer cell lines Hep3B or MCF7 were co-culturedwith fibroblasts and growth dynamics and interactions were investigated.Conditioned medium approaches and soft agar colony formation wereemployed to study the effects of soluble factors from Spalax, mice, andAcomys fibroblasts on cancer cell proliferation. Light microscopy andTransmission electron microscopy. For histological examination thesamples were fixed in paraformaldehyde, dehydrated, and embedded inparaffin. Sections were routinely stained with hematoxylin and eosin forpathological examination. For transmission electron microscopy specimenswere prepared as previously described (Manov et al. 2011).

Cell cycle analysis. The cell cycle distribution was assessed by flowcytometry of propidium iodide (PI)-stained nuclei.

Supplementary Information Detailed Materials and Methods

Animals: Spalax, spiny mice (Acomys cahirinus), rats (Ramis norregicus)and C57BL/6 mice were tested, Spalax and Acomys were captured in thefield and housed under ambient conditions in individual cages in theAnimal Facility of the Institute of Evolution, University of Haifa.Noteworthy, Spalax do not undergo uniform acclimatization upon transferfrom their natural habitat to a standardized laboratory environment butrather behave differentially according to their eco-geographic origins(Nevo 1999). The C57BL/6 mice were purchased from Harlan Laboratories(Jerusalem, Israel). Rats were supplied by the Animal House of thePsychology Department of Haifa University. All animals were kept withfree access to food and water at 21-23° C. in a 12:12 light-dark cycle.All animals used for experiments were healthy. Animals were sacrificedwith an inhalation anesthesia agent (isofluorane) overdose. All animalexperiments were approved by the Institutional Ethics Committee.

DMBA/TPA treatment: Four Spalax individuals of 2 year old and 4individuals over 10 years old; and 6 individuals of C57BL/6 mice, 3-4month old, were used in the 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) experiments.A single application of 200 μg of DMBA (Sigma Aldrich, Inc.) dissolvedin 100 μL of acetone for mice, and 500 μg of DMBA dissolved in 250 μLacetone for Spalax were used. The solution was applied onto the bareskin of the animal. Three days after the initial DMBA dose, mice weretreated with 30 μg of TPA (Sigma Aldrich, Inc.) dissolved in 100 uL ofacetone, and Spalax with 60 μg of TPA dissolved in 250 μL of acetone.TPA was applied 3 times per week for 2-3 months after which all micewere sacrificed upon skin carcinoma formation, and continued with Spalaxfor another 3 months twice a week.

3-MCA carcinogen treatment: 3-methylcholanthrene (3-MCA) has beencommonly used for induction of tumors in rodents (Malins et al. 2004).In this experimental system, mice and rats develop local fibrosarcomasin 2 to 4 months, respectively (Krelin et al. 2007) and can be palpatedas early as 30 to 60 days following injection. The recommended amount inthe literature of 3MCA (Sigma Aldrich, Inc.) treatment of mice is 200 μgdissolved in 200 μL, of olive oil. We calculated the amount applied torats and Spalax according to their average weight. Hence, animals weretreated with a single injection of 3MCA as follows: 200 μg/200 for mice;1 mg/500 μL for Spalax; and 1.5 mg/500 μL for rats. Animals used in thisexperiment were: six 2 years old Spalax individuals; six 10 years orolder Spalax individuals; six 3-4 months-old mice; and six 3 months oldwhite rats.

Animals were observed once a week until tumors could be palpated, andthen 2-3 times a week. Animals were sacrificed, tissues were removed,and either immediately frozen in Liquid-N₂ and kept at −80° C., or fixedin paraformaldehyde.

Cell culture: Primary Spalax, mice, and Acomys fibroblast cells wereisolated from under arm skin and lungs as described (Glaysher & Cree2011), and grown in DMEM-F12 medium (Biological Industries, Beit HaemeqIsrael), supplemented with 15% fetal bovine serum (FBS). Human cancercell lines Hep3B and MCF7 are commercially available, and were grown inRPMI (Hep3B) andDMEM (MCF7) supplemented with 10% MS, L-glutamine,penicillin and streptomycin (Biological Industries, Beit Haemeq Israel).Cells were incubated in a humidified atmosphere of 5% CO₂ and 95% O₂ at37° C. Spalax-derived fibrosarcoma cells were isolated from tumordeveloped after 3MCA injection. Tumor specimen was minced and treatedwith collagenase (1 mg/ml) under aseptic conditions to obtain asingle-cell suspension, which was plated in cultures dishes in DMEM-F12medium supplemented with 15% FBS andpenicillin-streptomycin-amphotericin B solution. Cells were seriallycultured more than 40 times.

Co-cultures of cancer cells and fibroblasts. Normal fibroblasts andhuman derived Hep3B cells were co-plated in 6-well plates in 2 ml ofculture medium Roswell Park Memorial Institute medium (RPMI)/Dulbecco'sModified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) (1:1)supplemented with 15% fetal bovine serum (FBS) (1:1) (BiologicalIndustries, Kibbutz Beit Haemeq, Israel). Fibroblasts were plated first(5×10⁴), and cancer cells were added within 1 h (5×10³), with a 10:1fibroblast-to-cancer cell ratio. In parallel, cultures of cancer cellsand. fibroblasts were plated with the same number of cells. The mediumwas changed every 3 days. Fibroblast-cancer cell co-interactions wereobserved and Photographed by using inverted microscopy.

Generation of conditioned medium (CM). Normal fibroblasts or cancercells (1×10⁶ cells) were plated in 10-cm tissue culture dishes andcultured in full medium containing 10% FBS for 4 days, thereaftersupernatants were collected and cells were removed by centrifugation.The cell-free CM was then diluted with fresh culture medium (1:1) andused for further experiments. To investigate the viability andproliferation rates of cancer cells exposed to CM, normal fibroblasts orcontol medium, we used PrestoBlue® dye reagent (invitrogen), asdescribed in (Manov et al. 2004), and trypan-blue standard treatmentfollowed by cell count using an automatic cell counter (Countess®, LifeTechnologies).

Soft agar colony formation assay was performed as described (Tyan el al.2011). In brief 2×10⁵ fibroblasts were seeded in 35-mm culture dishesand cultured for 2-3 days to reach confluence. After washing with PBS, 1ml of 0.5% agar in DMEM-F12 containing 2% FBS was added on top offibroblasts to form a base layer. After the agar was solidified, 5×10⁵MCF7 cells were suspended in 1 ml of 0.35% agar in DMEM containing 5%FBS and then added into the dish to form a cancer cell layer. Colonieswere stained with 0.05% crystal violet solution and counted under lightmicroscope after 5 and 11 days.

Microscopy: For histological examination the samples were fixed in 4%paraformaldehyde dissolved in PBS, dehydrated in increasingconcentrations of ethanol, and embedded in paraffin. Five-micrometersections were cut from paraffin blocks and routinely stained withhematoxylin and eosin for pathological examination.

For transmission electron microscopy specimens were fixed in 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), postfixedwith 2% OsO4, dehydrated in ethanol series and embedded in epoxy resin.Semi-thin sections were stained with 1% Toluidine Blue. Ultrathinsections (60 nm) were cut with a diamond knife, placed on 300-meshcopper grids, stained with 1% uranyl acetate, and viewed andphotographed with a Transmission Electron Microscope,

Cell cycle analysis: The cell cycle distribution was assessed by flowcytometry of propidium iodide (PI)-stained nuclei. Following incubation,cells were harvested by trypsin, combined with medium containingfloating cells, washed with PBS and stained with hypotonic PI solution(PI 50 μg/ml in 0.1% sodium citrate and 0.1% Triton X-100). The PIfluorescence of individual nuclei was recorded by FACSaria, (BectonDickinson, N.J., USA). A total of 10,000 events were acquired andcorrected for debris and aggregate population.

Example 1 Resistance of Spalax to Chemically-Induced Cancer

To assess experimentally if Spalax is resistant to chemically-inducedcarcinogenesis, we treated animals from different rodent speciesaccording to the following protocols:

DMBA/TPA treatment: Spalax and C57B/6 mice were treated with DMBA/TPA toinduce skin carcinogenesis (Goerttler et al. 1982). Spalax animalsdeveloped severe skin lesions within ten days (FIG. 1A, upper middlepanel). Histological examination of hematoxylin and eosin stained tissuesections demonstrated extensive skin necrosis involving the deep partsof the dermis, massive infiltration of the affected areas withneutrophil leukocytes, and ulcerated epidermis focally covered withfibrino-purulent exudates (FIG. 1A, lower middle panel). Thesubcutaneous skeletal muscle and bone tissues were not affected, and notumor was identified. The wounds completely healed within 7-9 weeks,resulting in epidermal thickening (FIG. 1A, right panels), and nofurther progression to skin tumors was observed, even though TPAtreatments were extended to 6 months (November 2010-April 2011). In thecontrol group, Spalax treated with acetone only did not show any changesin their skin macro- and microstructure, similar to non-treated animals(FIG. 1A, left panels). Following 7-10 days of DMBA/TPA treatment, micedemonstrated small intra-epidermal blisters; some of them rupturedforming superficial erosions with extensive crusting (FIG. 1B, middlepanels), which subsequently underwent malignant transformation anddevelopment of multiple skin tumors within 2 months (FIG. 1B, upperright panel). Histological examination revealed papillary and flatepidermal outgrowths with prominent dysplastic features, similar tosquamous cell carcinoma (FIG. 1B, right panels).

3-MCA treatment: the ability of a single subcutaneous 3-MCA injection toinduce fibrosarcoma is well documented (Krelin et al. 2007). Theexpected tumors appeared within 2-3 months in mice, and in 4-6 months inrats. Hypercellular spindle cell tumors with highly pleiomorphic,extensively proliferating cells (30 and more mitotic figures per 10 highpower fields) arranged into intersected bundles or wide sheets wereidentified. Scant, partially myxoid stroma and areas of hemorrhagicnecrosis were typical findings (FIG. 2, upper panel). While all examinedtumors which had been developed in 3-MCA-treated mice were qualified ashigh-grade fibrosarcomas, some of the rats presented lesions resemblinglow- to intermediate-grade fibrosarcoma. Importantly, Spalax did notshow any pathological process for over a year. However, by 14 to 16months following the 3-MCA treatment, two of the Spalax animals (out ofsix old individuals and a total of twelve animals) developed a tissueovergrowth at the site of the injection. These lesions were wellcircumscribed in shape, unlike the ill-defined tumors found in mice(FIG. 2, lower panel). Histological examination revealed benign spindlecell proliferation most probably reflecting fibrosis at the site ofincompletely resolved inflammatory reaction. Nonetheless, a single oldSpalax individual developed 3-MCA-induced cancer 18 month after initialtreatment (FIG. 3). A biopsy was performed, and the histologicalexamination revealed a partially necrotic and heavily inflamed, spindleand epithelioid cell tumor with infiltrative borders and myxoid stroma.Cells demonstrated dyscohesion, polymorphism in size and shape (bizarreand giant cells present) and prominent nuclear atypia (FIG. 3A). Thishypercellular tumor demonstrated high mitotic activity (above 30 mitosesper 10 high power fields) with abundant atypical mitotic figuresTransmission electron microscopy revealed fibrosarcoma-like findings(Antonescu & Wren 2004): deformed nuclei, some with monstrousappearance; long branching and dilated rough endoplasinic reticulum andabundance of extracellular collagen fibers (FIGS. 3B and 3C).Myofibroblastic differentiation was not observed. A cancer cell line wasestablished from the tumor sample. Cells, able to attach and survive,showed a typical fibroblast phenotype (FIG. 3D). The morphology ofisolated cells remained unchanged throughout a long culture time (40passages, 8 months after isolation).

The remaining treated Spalax individuals showed no phenotypic orbehavioral changes, and are still under observation in the Animal Houseover two years following treatment (October 2010-January 2013).

Example 2 Spalax Fibroblasts Suppress Growth of Cancer Cells In Vitro

To compare the effects of Spalax and mouse fibroblasts on growth ofhuman epithelial cancer (Hep3B), we used a co-culture approach, whereskin fibroblasts isolated from newborn rodents were cultured togetherwith cancer cells on a shared surface (FIG. 4). The number of cancercells co-cultured with mouse fibroblasts increased gradually, and on Day7, Hep3B cells surrounded by mouse fibroblasts reached 80% confluence,similar to control (Hep3B only). In contrast, obvious inhibition ofcancer cell growth was found when Hep3B cells were co-cultured withSpalax fibroblasts: foci of destroyed cancer cells were visible (FIG.4). Prolonged co-cultivation up to 11 days resulted in furtherdestruction of cancer cell colonies by Spalax fibroblasts and the spacespreviously occupied by Hep3B cells were invaded by fibroblasts.Overgrown Hep3B colonies were found when co-cultured with mousefibroblasts.

Since we compare a wild mammal with laboratory animals that could besensitive to cancer, we conducted a series of experiments using skinfibroblasts isolated from Acomys, which like Spalax, is a wild rodent,however with a short life span. As shown in FIG. 5, no inhibitory effectwas found when Acomys fibroblasts were co-cultured with Hep3B cells. Onthe contrary, co-culture with Acomys fibroblasts promoted cancer cellinvasion similar to mouse fibroblasts.

Next, we studied the cancer-suppressing ability of Spalax fibroblastsisolated from lung tissues of newborn animals, to confirm that cancerinhibition is not limited to skin fibroblasts (FIG. 5), Hep3B colonieswere similarly inhibited and destroyed when co-cultured with Spalax lungfibroblasts.

To determine whether the anti-cancer activity of Spalax fibroblasts wasmediated by fibroblast-secreted soluble factors, conditioned medium andsoft agar colony formation approaches were used. Cancer cells ofdifferent origins were incubated under conditioned media of normalfibroblasts, which had not been previously exposed to cancer cells orother stimuli. As demonstrated in FIG. 6A, exposure of Hep3B cells toconditioned medium from cultured Spalax fibroblasts decreased cancercell growth, while mouse fibroblast conditioned medium had no effect.Importantly, conditioned medium generated by Spalax normal fibroblastsgradually suppressed growth of the homologous tumor, Spalax-derivedfibrosarcoma (FIG. 6B). Further, we evaluated the ability of fibroblastsisolated from Spalax or mouse to promote or inhibit cancer cell colonyformation in soft agar. Remarkably more colonies were formed when humanMCF7 cells were co-cultured with mouse fibroblasts compared to Spalax(FIG. 6C). In contrast, a monolayer of Spalax fibroblasts reduced MCF7colony-formation (compared to control).

To validate that the cancer-inhibiting effect is not limited tofibroblasts from newborn individuals, we isolated skin fibroblasts fromadult Spalax (>5.5 years old). The ability of conditioned mediumgenerated by adult Spalax fibroblasts to kill Hep3B cells is presentedin FIG. 6D. Following 9 days of exposure, both floating and attachedcells were collected and their survival rates were determined by atrypan blue extrusion assay. Only 51% of cancer cells incubated underSpalax fibroblasts conditioned medium survived while 93% of Hep3B cellsincubated in self-conditioned medium (or mice conditioned media)remained adherent and viable (FIG. 6E).

We further examined whether Spalax fibroblast conditioned medium affectsthe growth of non-cancerous cells. No inhibitory effect was found whenmouse and Spalax normal fibroblasts were exposed to conditioned mediumgenerated by Spalax fibroblasts (FIG. 6F).

Like Spalax, naked mole rat fibroblasts also demonstrate anticanceractivity presented as destroyed cancer cells when co-cultured togetherwith the naked mole rat's fibroblasts (FIG. 7).

To investigate the mechanisms by which Spalax fibroblasts induce cancercell death, we examined the cell cycle distributions in Hep3B. Nochanges in the cell cycle distribution were found when Hep3B cells wereincubated with CM generated by immortalized cell line generated from thesame fibroblast cancer-affecting primary cells, or when compared toHep3B grown with their own medium (FIG. 8). In contrast, followingexposure to ,Spalax CM, Hep3B cell cycle analysis revealed a noticeableaccumulation of dead cells in sub-G1 (approx. 30%).

Migration assay revealed that Spalax normal fibroblasts did not promotethe migration of breast cancer cells MDA-MB-231 through a porousmembrane (Transwelt, Corning Inc.). In contrast, immortalized cellspromoted the invasion of cancer cells towards fibroblasts (FIG. 9).

REFERENCES

-   Antonescu C R, Garen A (2004). Spectrum of low-grade fibrosarcomas:    a comparative ultrastructural analysis of low-grade myxofibrosarcoma    and fibromyxoid sarcoma. Ultrastructural pathology 28, 321-332.-   Avivi A, Ashur-Fabian O, Joel A, Trakhtenbrot L, Adamsky K,    Goldstein I, Amariglio N, Rechavi G , Nevo E (2007). P53 in blind    subterranean mole rats-loss-of-function versus gain-of-function    activities on newly cloned Spalax target genes. Oncogene. 26,    2507-2512.-   Avivi A, Gerlach F, Joel A, Reuss S, Burmester T, Nevo E , Hankeln T    (2010). Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia    adaptation of the subterranean mole rat Spalax. Proceedings ofthe    National Academy of Sciences of the United States of America. 107,    21570-21575.-   Avivi A, Shams I, Joel A, Lache O, Levy A P , Nevo E (2005).    Increased blood vessel density provides the mole rat physiological    tolerance to its hypoxic subterranean habitat. Faseb J. 19,    1314-1316.-   Flesher J W, Horn J Lehner A F (1998). Carcinogenicity of    1-hydroxy-3-methylcholanthrene and its electrophilic sulfate ester    1-sulfooxy-3-methylcholanthrene in Sprague-Dawley rats. Biochem    Biophys Res Cominun. 243, 30-35.-   Glaysher S Cree I A (2011). Isolation and culture of colon cancer    cells and cell lines. In Cancer Cell Culture. (I A Cree, ed).    Portsmouth, UK: Humana Press, pp. 135-140.-   Goettler K., Loehrke H, Hesse B , Schweizer J (1984). Skin tumor    formation in the European hamster (Cricetus cricetus L.) after    topical initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and    promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA).    Carcinogenesis. 5, 521-524.-   Goettler K., Loehrke H, Schweizer J , Hesse B (1982). Diterpene    ester-mediated two-stage carcinogenesis, Carcinogenesis: a    comprehensive survey. 7, 75-83.-   Izzotti A, Camoirano A, Cartiglia C, Grubbs C J, Lubet R A, KeHoff G    J, De Flora S (1999). Patterns of DNA adduct formation in liver and    mammary epithelial cells of rats treated with    7,12-dimethyibenz(a)anthra.cene, and selective effects of    chemopreventive agents. Cancer research. 59, 4285-4290.-   Krelin Y, Voronov E, Dotan S, Eikabets M, Reich E, Fogel M., Huszar    M, Iwakura Y, Segal S, Dinarello C A, Apte R N (2007), interleukin-1    beta-driven inflammation promotes the development and invasiveness    of chemical carcinogen-induced tumors. Cancer research. 67,    1062-1071.-   Malik A, Komi A, Hubner S, Hernandez A G, Thimmapuram J, Ali S,    Glaser F, Paz A, Avivi A , Band M (2011). Transcriptome sequencing    of the blind subterranean mole rat, Spalax galili: utility and    potential for the discovery of novel evolutionary patterns. PloS    one. 6, e21227.-   Malik A, Korol A, Weber M, Hankeln T, Avivi A , Band M (2012).    Transcriptome analysis of the spalax hypoxia survival response    includes suppression of apoptosis and tight control of angiogenesis.    BMC genomics. 13, 615.-   Malins D C, Anderson K M, Gilman N K, Green V M, Barker E A    Hellstrom K E (2004). Development of a cancer DNA phenotype prior to    tumor formation. Proceedings of the National Academy of Sciences of    the United States of America. 101, 10721-10725.-   Manov I, Hirsh M Iancu I C (2004). N-acetylcysteine does not protect    HepG2 cells against acetaminophen-induced apoptosis. Basic Clin    Pharmacol Toxicol. 94, 213-225.-   Manov I, Hirsh M, Iancu I C, Malik A, Sotnichenko N, Band M, Avivi A    , Shams I (2013). Pronounced cancer resistance in a subterranean    rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.    BMC biology. 11, 91.-   Manov I, Pollak Y, Broneshter R, Iancu I C (2011). Inhibition of    doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B    cells by sorafenib—the role of extracellular signal-regulated kinase    counteraction. FEBS J. 278, 3494-3507.-   Nasser N J, Avivi A, Shafat I, Edovitsky E, Zcharia E, Ilan N,    Vlodaysky Nevo E (2009). Alternatively spliced Spalax heparinase    inhibits extracellular matrix degradation, tumor growth, and    metastasis. Proceedings of the National Academy of Sciences of the    United States of America, 106, 2253-2258.-   Nevo E (1999). Mosaic Evolution of Subterranean Mammals: Regression,    Progression and Global Convergence. Oxford: Oxford University Press.-   Nevo E, Ivanitskaya E Beiles A (2001). Adaptive Radiation of Blind    Subterranean Mole Rats. Leiden: Backhuys.-   Ravid O, Shams I, Ben Califa N, Nevo E, Avivi A, Neumann D (2007).    An extracellular region of the erythropoietin receptor of the    subterranean blind mole rat Spalax enhances receptor maturation.    Proceedings of the National Academy of Sciences of the United States    of America. 104, 14360-14365.-   Schulke S, Dreidax D, Malik A, Burmester T, Nevo E, Band M, Avivi A    , Hankeln T (2012). Living with stress: Regulation of antioxidant    defense genes in the subterranean, hypoxia-tolerant mole rat,    Spalax. Gene.-   Shams I, Avivi A, Nevo E (2005a). Oxygen and carbon dioxide    fluctuations in burrows of subterranean blind mole rats indicate    tolerance to hypoxic-hypercapnic stresses. Comp Biochem Physiol A    Mol Integr Physiol. 142, 376-382.-   Shams I, Nevo E , Avivi A (2005b). Ontogenetic expression of    erythropoietin and hypoxia-inducible factor-1 alpha genes in    subterranean blind mole rats. Faseb J. 19, 307-309.-   Tyan S W, Kuo W H, Huang C K, Pan C C, Shew J Y, Chang K J, Lee E Y    Lee W H (2011). Breast cancer cells induce cancer-associated    fibroblasts to secrete hepatocyte growth factor to enhance breast    tumorigenesis. PloS one. 6, e15313.-   Yuspa S H (1998). The pathogenesis of squamous cell cancer: lessons    learned from studies of skin carcinogenesis. Journal of    dermatological science. 17, 1-7.

1. A method for treatment of cancer or inhibiting cancer metastasis in ahuman subject diagnosed with cancer, said method comprisingadministering to said subject an effective amount of a pharmaceuticalcomposition comprising a conditioned cell culture medium of healthycells derived from Spalax or naked mole rat (Heterocephalus glaber), anda pharmaceutically active carrier, excipient or diluent.
 2. The methodaccording to claim 1, wherein said cells are derived from Spalaxehrenbeigi (Nannospalax).
 3. The method according to claim 1, whereinsaid cells are skin or lung fibroblasts.
 4. The method according toclaim 3, wherein said fibroblasts are primary cells.
 5. The methodaccording to claim I. wherein said cancer is selected from the groupconsisting of carcinoma, sarcoma, glioma, leukemia and lymphoma.
 6. Themethod according to claim 5, wherein said carcinoma is selected from thegroup consisting of breast cancer carcinoma, hepatocellular carcinomaand breast adenocarcinoma.
 7. The method according to claim 1, furtherincluding the concomitant administration of at least onechemotherapeutic agent.
 8. The method according to claim 1, wherein saidcomposition further comprises at least one chemotherapeutic agent.
 9. Amethod for killing cancer cells, inhibiting proliferation of cancercells or inhibiting cancer cell migration, comprising administering to ahuman subject in need thereof an effective amount of a pharmaceuticalcomposition comprising a conditioned cell culture medium of healthycells derived from Spalax or naked mole rat (Heterocephalus glaber), anda pharmaceutically active carrier, excipient or diluent.
 10. The methodaccording to claim 9, wherein said cells are derived from Spalaxehrenbergi (Nannospalax).
 11. The method according to claim 9, whereinsaid cells are skin or lung fibroblasts.
 12. The method according toclaim 11, wherein said fibroblasts are primary cells.
 13. The methodaccording to claim 9, wherein said cancer cells are selected from thegroup consisting of carcinoma cells, sarcoma cells, glioma cells,leukemia cells and lymphoma cells.
 14. The method according to claim 13,wherein said carcinoma cells are selected from the group consisting ofbreast cancer carcinoma cells, hepatocellular carcinoma cells and breastadenocarcinoma cells.
 15. The method according to claim 9, furtherincluding the concomitant administration of at least onechemotherapeutic agent.
 16. The method according to claim 9, whereinsaid composition further comprises at east one chemotherapeutic agent.